In man, responses to HLA antigens dominate the strong immunological reaction to transplanted tissue. In order to avoid rejection of transplanted tissue, it is desirable to match donor HLA type to the recipient HLA type. The HLA type of an individual is determined by antigens encoded by genes on a single chromosome. Four principal HLA antigen loci have been identified on chromosome six and have been designated A, B, C and D (and the closely-related DR locus). The major lymphocyte activating determinants are controlled by alleles at the forth locus, D. The matching of recipient and donor D allotypes has proved to be a major factor in reducing transplant rejection and transfusion reaction. This is most likely attributable to the fact that cytotoxic T lymphocytes against HLA A and B antigens are generated by D locus differences.
Typing of the D locus is typically carried out in a one-way mixed lymphocyte reaction (MLR). In a MLR, two groups of genetically dissimilar lymphocytes are cultured together. One of the populations (stimulators) has been made immunologically unresponsive by treatment with radiation or Mitomycin C. The other cell population responds to the phenotypically dissimilar determinants on the surface of the irradiated cell population. To conduct a typing assay employing MLR, a screening panel of donors with known HLA D types is required as stimulators. This panel of homozygous typing cells are best found in families of cousin marriages. The unknown donor's PBL are cultured individually with the typing panel for five to seven days. If the unknown donor's HLA D allotype is different than the allotype of the screening lymphocyte, there will be an immunological response by the donor's PBL. The unknown donor's allotype will be indicated, therefore, by nonreactivity in the culture containing the stimulating lymphocytes of identical HLA D type.
Another method employed to determine HLA D type is the mixed lymphocyte cytotoxcity test (MLC). The first phase of the MLC requires the stimulating of the donor's lymphocytes with a screening panel as described above for the MLR (5-7 days). Responding primed lymphocytes are harvested and assayed for cytotoxcity on a panel of B lymphoblastoid cell lines with known HLA D types. These target cells have been prelabeled with a radioisotope. Donor lymphocytes from the first phase, which have been primed to a foreign HLA D type, will kill the radiolabeled targets with the same D type to which they have been primed. Cytotoxic reaction can be detected by radioactivity in the supernatant. This assay requires an additional 6 to 8 hours after the MLR phase.
While the above methods are accurate to determine HLA D type, the lengthy procedures involved are unsuitable for typing donors and recipients for transplant procedures. Generally, the donor is a cadaver and it is necessary to transplant the organ as quickly as possible (e.g., within 24 hours). A need, therefore, exists for an expedient test for HLA D type.